Collagen-targeted nanoparticles

ABSTRACT

This invention relates to compositions comprising collagen binding peptides coupled to nanoparticles. The invention also relates to a method of imaging a collagenous matrix using a composition comprising collagen binding peptides coupled to nanoparticles.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/000,036, filed Aug. 16, 2013, now U.S. Pat. No. 9,173,919, which is anational stage entry under 35 U.S.C. §371(b) of InternationalApplication No. PCT/US2012/025431, filed Feb. 16, 2012, which claims thebenefit under 35 U.S.C. §119(e) of U.S. Provisional Application No.61/443,572, filed Feb. 16, 2011, and U.S. Provisional Application No.61/488,385, filed May 20, 2011, the entire disclosures of which arehereby incorporated by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 30, 2014, isnamed 3220E-226330_SL.txt and is 9,221 bytes in size.

TECHNICAL FIELD

This invention generally pertains to the field of nanomedicine. Moreparticularly, the invention pertains to collagen-binding peptidescoupled to nanoparticles.

BACKGROUND AND SUMMARY OF THE INVENTION

Nanoparticles are submicron materials that often possess differentproperties than bulk material of the same kind. Nanoparticles have beenstudied for uses in many fields, including diagnostic and therapeuticapplications in the life sciences. Because of their small size andunique properties, nanoparticles often have enhanced distribution in thebody compared to larger sized particles. Further, nanoparticles may bespecifically directed to particular targets in the body by attaching oneor more components to the nanoparticle surface (i.e. functionalization).Functionalization of a nanoparticle with a component having affinity fora specific target in the body, for example, collagen, can direct thenanoparticle to tissues containing the target molecule.

There are more than 20 types of collagen currently identified, with typeI being the most common. Many tissues are composed primarily of type Icollagen including tendon, ligament, skin, and bone. While each of thesestructures also contains other collagen types, proteoglycans andglycosaminoglycans, and minerals in the case of bone, the principlecomponent is type I collagen.

Disclosed herein is targeted delivery of nanoparticles, utilizingcollagen as an attachment site for functionalized nanoparticles.Collagens are a component of the extracellular matrix (ECM), which isresponsible for supporting cells, thus making up connective tissues suchas blood vessels, cartilage, or skin. Collagens and their correspondingbinding domains have been well studied. Collagen binding domains can bereduced to specific peptide sequences, which bind specifically andexclusively to collagen, the target protein. Utilizing peptidesynthesis, it is possible to immobilize a collagen-binding peptide, forexample a collagen type I or type II binding peptide, ontonanoparticles, allowing those nanoparticles to fasten specifically totype I or type II collagen, respectively.

This targeted delivery system is not limited to type I collagen, as manyother collagen types have also been defined. As such, specific targetingof a variety connective tissues and proteins is also contemplated.Collagen-binding gold nanoparticles, for example, are useful for avariety of biomedical imaging techniques including SEM, TEM, confocalmicroscopy, MRI, and photoacoustic imaging. Further, radiopaquenanoparticles that are coupled with collagen binding proteins can beused in radiologic imaging such as CT scan or X-ray.

The following various embodiments are contemplated:

1) A composition comprising at least one collagen binding polypeptidecoupled to a nanoparticle, wherein the polypeptide contains from 7 aminoacids to 40 amino acids and wherein the polypeptide does not form atriple helix.

2) The composition of clause 1 wherein the nanoparticle furthercomprises a stabilizer.

3) The composition of clause 2 wherein the stabilizer is selected fromthe group consisting of a polyethylene glycol (PEG), a dextran, apeptide, an alkane-thiol, and an oligonucleotide-thiol.

4) The composition of clause 3 wherein the stabilizer is PEG.

5) The composition of any of clauses 1 to 4 wherein the polypeptidecontains from 9 amino acids to 40 amino acids.

6) The composition of any of clauses 1 to 4 wherein the polypeptidecontains from 9 amino acids to 30 amino acids.

7) The composition of any of clauses 1 to 4 wherein the polypeptidecontains from 9 amino acids to 18 amino acids.

8) The composition of any of clauses 1 to 4 wherein the polypeptide isselected from the group consisting of RRANAALKAGELYKSILY (SEQ ID NO: 1),RRANAALKAGELYKCILY (SEQ ID NO: 2), GELYKSILY (SEQ ID NO: 3), GELYKCILY(SEQ ID NO: 4), SYIRIADTNIT (SEQ ID NO: 5), TKKTLRT (SEQ ID NO: 6),SQNPVQP (SEQ ID NO: 7), RLDGNEIKR (SEQ ID NO: 8), KELNVYT (SEQ ID NO:9), KLWVLPK (SEQ ID NO: 10), CQDSETRTFY (SEQ ID NO: 11), AHEEISTTNEGVM(SEQ ID NO: 12), GLRSKSKKFRRPDIQYPDATDEDITSHM (SEQ ID NO: 13),GSITTIDVPWNV (SEQ ID NO: 14), and NGVFKYRPRYFLYKHAYFYPPLKRFPVQ (SEQ IDNO: 15).

9) The composition of any of clauses 1 to 4 wherein the polypeptide isselected from the group consisting of RRANAALKAGELYKSILY (SEQ ID NO: 1),RRANAALKAGELYKCILY (SEQ ID NO: 2), GELYKSILY (SEQ ID NO: 3), GELYKCILY(SEQ ID NO: 4), SYIRIADTNIT (SEQ ID NO: 5), TKKTLRT (SEQ ID NO: 6),SQNPVQP (SEQ ID NO: 7), and RLDGNEIKR (SEQ ID NO: 8).

10) The composition of any of clauses 1 to 4 wherein the polypeptidecomprises the amino acid sequence GELYKXILY, wherein X is serine,cysteine, or threonine (SEQ ID NO: 16).

11) The composition of any of clauses 1 to 4 wherein the polypeptide isRRANAALKAGELYKSILY (SEQ ID NO: 1).

12) The composition of any of clauses 1 to 4 wherein the polypeptide isRRANAALKAGELYKCILY (SEQ ID NO: 2).

13) The composition of any of clauses 1 to 12 further comprising acysteine at the amino terminal region or carboxy terminal region.

14) The composition of clause 13 further comprising a spacer.

15) The composition of clause 14 wherein the spacer comprises at leastone glycine.

16) The composition of any of clauses 1 to 12 further comprising apeptide sequence selected from the group consisting of GC, CG, and GCG.

17) The composition of any of clauses 1 to 16 wherein the nanoparticleis a polymeric nanoparticle, a metallic nanoparticle, a semiconductornanoparticle, or any combination thereof.

18) The composition of clause 17 wherein the nanoparticle is a goldnanoparticle.

19) The composition of clause 17 wherein the nanoparticle is an ironoxide nanoparticle.

20) The composition of any of clauses 1 to 19 further comprising acarrier.

21) The composition of clause 20 wherein the carrier is apharmaceutically acceptable carrier.

22) The composition of clause 21 wherein the carrier is a liquid carrierand is selected from the group consisting of saline, glucose, alcohols,glycols, esters, amides, and a combination thereof.

23) An effective dose of the composition of any of clauses 1 to 22 foradministration to a patient, wherein the effective dose ranges fromabout 1 ng to about 1 mg per kilogram of body weight.

24) An effective dose of the composition of any of clauses 1 to 22 foradministration to a patient, wherein the effective dose ranges fromabout 1 pg to about 10 ng per kilogram of body weight.

25) An effective dose of the composition of any of clauses 1 to 22 foradministration to a patient, wherein the effective dose ranges fromabout 1 μg to about 100 μg per kilogram of body weight.

26) A composition for use in imaging a collagenous matrix, thecomposition comprising at least one collagen binding polypeptide coupledto a nanoparticle, wherein the polypeptide contains from 7 amino acidsto 40 amino acids and wherein the polypeptide does not form a triplehelix.

27) The composition of clause 26 wherein the nanoparticle furthercomprises a stabilizer.

28) The composition of clause 27 wherein the stabilizer is selected fromthe group consisting of a polyethylene glycol (PEG), a dextran, apeptide, an alkane-thiol, and an oligonucleotide-thiol.

29) The composition of clause 28 wherein the stabilizer is PEG.

30) The composition of any of clauses 26 to 29 wherein the polypeptidecontains from 9 amino acids to 40 amino acids.

31) The composition of any of clauses 26 to 29 wherein the polypeptidecontains from 9 amino acids to 30 amino acids.

32) The composition of any of clauses 26 to 29 wherein the polypeptidecontains from 9 amino acids to 18 amino acids.

33) The composition of any of clauses 26 to 29 wherein the polypeptideis selected from the group consisting of RRANAALKAGELYKSILY (SEQ ID NO:1), RRANAALKAGELYKCILY (SEQ ID NO: 2), GELYKSILY (SEQ ID NO: 3),GELYKCILY (SEQ ID NO: 4), SYIRIADTNIT (SEQ ID NO: 5), TKKTLRT (SEQ IDNO: 6), SQNPVQP (SEQ ID NO: 7), RLDGNEIKR (SEQ ID NO: 8), KELNVYT (SEQID NO: 9), KLWVLPK (SEQ ID NO: 10), CQDSETRTFY (SEQ ID NO: 11),AHEEISTTNEGVM (SEQ ID NO: 12), GLRSKSKKFRRPDIQYPDATDEDITSHM (SEQ ID NO:13), GSITTIDVPWNV (SEQ ID NO: 14), and NGVFKYRPRYFLYKHAYFYPPLKRFPVQ (SEQID NO: 15).

34) The composition of any of clauses 26 to 29 wherein the polypeptideis selected from the group consisting of RRANAALKAGELYKSILY (SEQ ID NO:1), RRANAALKAGELYKCILY (SEQ ID NO: 2), GELYKSILY (SEQ ID NO: 3),GELYKCILY (SEQ ID NO: 4), SYIRIADTNIT (SEQ ID NO: 5), TKKTLRT (SEQ IDNO: 6), SQNPVQP (SEQ ID NO: 7), and RLDGNEIKR (SEQ ID NO: 8).

35) The composition of any of clauses 26 to 29 wherein the polypeptidecomprises the amino acid sequence GELYKXILY, wherein X is serine,cysteine, or threonine (SEQ ID NO: 16).

36) The composition of any of clauses 26 to 29 wherein the polypeptideis RRANAALKAGELYKSILY (SEQ ID NO: 1).

37) The composition of any of clauses 26 to 29 wherein the polypeptideis RRANAALKAGELYKCILY (SEQ ID NO: 2).

38) The composition of any of clauses 26 to 37 further comprising acysteine at the amino terminal region or carboxy terminal region.

39) The composition of clause 38 further comprising a spacer.

40) The composition of clause 39 wherein the spacer comprises at leastone glycine.

41) The composition of any of clauses 26 to 37 further comprising apeptide sequence selected from the group consisting of GC, CG, and GCG.

42) The composition of any of clauses 26 to 41 wherein the nanoparticleis a polymeric nanoparticle, a metallic nanoparticle, a semiconductornanoparticle, or any combination thereof.

43) The composition of clause 42 wherein the nanoparticle is a goldnanoparticle.

44) The composition of clause 42 wherein the nanoparticle is an ironoxide nanoparticle.

45) The composition of any of clauses 26 to 44 further comprising acarrier.

46) The composition of clause 45 wherein the carrier is apharmaceutically acceptable carrier.

47) The composition of clause 46 wherein the carrier is a liquid carrierand is selected from the group consisting of saline, glucose, alcohols,glycols, esters, amides, and a combination thereof.

48) An effective dose of the composition of any of clauses 26 to 47 foradministration to a patient, wherein the effective dose ranges fromabout 1 ng to about 1 mg per kilogram of body weight.

49) An effective dose of the composition of any of clauses 26 to 47 foradministration to a patient, wherein the effective dose ranges fromabout 1 pg to about 10 ng per kilogram of body weight.

50) An effective dose of the composition of any of clauses 26 to 47 foradministration to a patient, wherein the effective dose ranges fromabout 1 μg to about 100 μg per kilogram of body weight.

51) A method for imaging a collagenous matrix, the method comprising thesteps of contacting a collagenous matrix with a composition comprisingat least one collagen binding polypeptide coupled to a nanoparticle,wherein the polypeptide contains from 7 amino acids to 40 amino acidsand wherein the polypeptide does not form a triple helix, and imagingthe collagenous matrix.

52) The method of clause 51 wherein the nanoparticle further comprises astabilizer.

53) The method of clause 52 wherein the stabilizer is selected from thegroup consisting of a polyethylene glycol (PEG), a dextran, a peptide,an alkane-thiol, and an oligonucleotide-thiol.

54) The method of clause 53 wherein the stabilizer is PEG.

55) The method of any of clauses 51 to 54 wherein the polypeptidecontains from 9 amino acids to 40 amino acids.

56) The method of any of clauses 51 to 54 wherein the polypeptidecontains from 9 amino acids to 30 amino acids.

57) The method of any of clauses 51 to 54 wherein the polypeptidecontains from 9 amino acids to 18 amino acids.

58) The method of any of clauses 51 to 54 wherein the polypeptide isselected from the group consisting of RRANAALKAGELYKSILY (SEQ ID NO: 1),RRANAALKAGELYKCILY (SEQ ID NO: 2), GELYKSILY (SEQ ID NO: 3), GELYKCILY(SEQ ID NO: 4), SYIRIADTNIT (SEQ ID NO: 5), TKKTLRT (SEQ ID NO: 6),SQNPVQP (SEQ ID NO: 7), RLDGNEIKR (SEQ ID NO: 8), KELNVYT (SEQ ID NO:9), KLWVLPK (SEQ ID NO: 10), CQDSETRTFY (SEQ ID NO: 11), AHEEISTTNEGVM(SEQ ID NO: 12), GLRSKSKKFRRPDIQYPDATDEDITSHM (SEQ ID NO: 13),GSITTIDVPWNV (SEQ ID NO: 14), and NGVFKYRPRYFLYKHAYFYPPLKRFPVQ (SEQ IDNO: 15).

59) The method of any of clauses 51 to 54 wherein the polypeptide isselected from the group consisting of RRANAALKAGELYKSILY (SEQ ID NO: 1),RRANAALKAGELYKCILY (SEQ ID NO: 2), GELYKSILY (SEQ ID NO: 3), GELYKCILY(SEQ ID NO: 4), SYIRIADTNIT (SEQ ID NO: 5), TKKTLRT (SEQ ID NO: 6),SQNPVQP (SEQ ID NO: 7), and RLDGNEIKR (SEQ ID NO: 8).

60) The method of any of clauses 51 to 54 wherein the polypeptidecomprises the amino acid sequence GELYKXILY, wherein X is serine,cysteine, or threonine (SEQ ID NO: 16).

61) The method of any of clauses 51 to 54 wherein the polypeptide isRRANAALKAGELYKSILY (SEQ ID NO: 1).

62) The method of any of clauses 51 to 54 wherein the polypeptide isRRANAALKAGELYKCILY (SEQ ID NO: 2).

63) The composition of any of clauses 51 to 62 further comprising acysteine at the amino terminal region or carboxy terminal region.

64) The composition of clause 63 further comprising a spacer.

65) The composition of clause 64 wherein the spacer comprises at leastone glycine.

66) The composition of any of clauses 51 to 62 further comprising apeptide sequence selected from the group consisting of GC, CG, and GCG.

67) The method of any of clauses 51 to 66 wherein the nanoparticle is apolymeric nanoparticle, a metallic nanoparticle, a semiconductornanoparticle, or any combination thereof.

68) The method of clause 67 wherein the nanoparticle is a goldnanoparticle.

69) The method of clause 67 wherein the nanoparticle is an iron oxidenanoparticle.

70) The method of any of clauses 51 to 69 further comprising a carrier.

71) The method of clause 70 wherein the carrier is a pharmaceuticallyacceptable carrier.

72) The method of clause 71 wherein the carrier is a liquid carrier andthe liquid carrier is selected from the group consisting of saline,glucose, alcohols, glycols, esters, amides, and a combination thereof.

73) The method of any of clauses 51 to 72 wherein an effective dose isadministered to a patient, the effective dose ranging from about 1 ng toabout 1 mg per kilogram of body weight.

74) The method of any of clauses 51 to 72 wherein an effective dose isadministered to a patient, the effective dose ranging from about 1 pg toabout 10 ng per kilogram of body weight.

75) The method of any of clauses 51 to 72 wherein an effective dose isadministered to a patient, the effective dose ranging from about 1 μg toabout 100 μg per kilogram of body weight.

76) The composition or method of any of clauses 1 to 75 wherein thepolypeptide is coupled to the nanoparticle using a crosslinking agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Stability of functionalized nanoparticles (NP). NPfunctionalized with polyethylene glycol (PEG) are stable in 1×PBS asshown by the absorbance spectrum in which the maximum absorbance appearsaround 525 nm and immediately drops off as in the NP control. Likewise,after coupling SILY (SEQ ID NO: 17) to the PEG functionalized NP,stability is maintained. There is a slight shift in maximum absorbancefrom 522 nm to approximately 530 nm which indicates an increase inparticle size consistent with coupling of PEG and SILY (SEQ ID NO: 17).FIG. 1 discloses ‘SILY’ as SEQ ID NO: 17.

FIG. 2. Binding of functionalized nanoparticles to collagen. NPfunctionalized with 2 kDa PEG showed increased binding to collagencompared to 5 kDa PEG. This is due to the length of the peptide SILY(SEQ ID NO: 17), which is 2197 Da and is masked in part by the highermolecular weight PEG. Minimal nonspecific binding to BSA was observed inboth functionalized nanoparticles.

FIG. 3. Nanoparticles localized on rat tail tendon. Tendons wereincubated with gold nanoparticles functionalized with either SILYpeptide (SEQ ID NO: 17) or PEG control. In A., GNP-SILY (‘SILY’disclosed as SEQ ID NO: 17) 60 nm spheres can be seen on the surface ofcollagen fibers in contrast to B. which was treated with GNP-PEG controlspheres. A closer image in C. shows GNP-SILY (‘SILY’ disclosed as SEQ IDNO: 17) binding along D-banded collagen fibers where it appears to bindin the overlap zone close to the following gap region. In all images,scale bar=1 μm.

FIG. 4. Schematic of the synthesis of PLGA core+pNIPAM shellnanoparticles (A and B) and the subsequent chemistry (C) used to targetthe nanoparticles to collagen type II. FIG. 4 discloses ‘WYRGRLGC’ asSEQ ID NO: 20.

FIG. 5. Transmission electron microscope images of the variousnanoparticles both pre- and post-lyophilization. Scale bars=250 nm.

FIG. 6. Temperature sweeps of the various nanoparticles. Data isrepresented as mean±standard deviation (n=3). C=PLGA core only; C+S#%=PLGA core+pNIPAM shell+mol % acrylic acid; L=Lyophilized.

FIG. 7. Toxicity of the core+shell nanoparticles in human monocytes, φrepresents p<0.05 compared to all other treatments (One-way ANOVA+Tukeypost-hoc test). Data is presented as mean±standard deviation (n=4).

FIG. 8. TNF-α production by human monocytes treated with core+shellnanoparticles. φ represents statistical significance from all other datapoints (p<0.05, One-way ANOVA+Tukey Post-hoc test). Data presented asmean±standard deviation (n=4).

FIG. 9. Collagen Type II binding assay for C+S nanoparticles. DifferentGreek letters represent statistical significance (p<0.05; One-wayANOVA+Tukey Post-hoc test). Data presented as mean±standard deviation(n=4).

DETAILED DESCRIPTION OF THE ILLUSTRATIVE EMBODIMENTS

As used herein, a “collagen binding peptide” means a polypeptide thatbinds to one or more types of collagen.

As used herein the terms “nanoparticle coupled collagen binding peptide”and “collagen binding peptide(s) coupled to a nanoparticle(s)” are usedinterchangeably and refer to one or more collagen binding peptidescoupled to one or more nanoparticles.

As used herein “collagenous matrix” refers to a tissue or componentthereof that contains one or more types of collagen.

A nanoparticle is understood by those of skill in the art to refer to aparticle having at least one dimension of submicron size. Nanoparticlesmay be composed from one or more of several types of materials, forexample polymers [e.g., Poly(lactic-co-glycolic acid) (PLGA) orpoly(N-isopropylacrylamide) (pNIPAM)], metals, semiconductors, and thelike. Exemplary nanoparticles are gold nanoparticles and iron oxidenanoparticles. For a review see Garg et al. (2007) PharmaceuticalReviews 5(6), available atwww.pharmainfo.net/reviews/nanoparticles-review, incorporated herein byreference.

The nanoparticles may also be composed of a combination of materialtypes, for example, as a core/shell structure. Core/shell nanoparticlesare nanostructures that have a core made of a material coated withanother material. For review of core/shell nanostructures see Zhang etal. (2008) Recent Patents on Biomedical Engineering 1:34-42,incorporated herein by reference. Illustratively, a core/shellnanoparticle may have a core and shell comprising one or more of severaltypes of materials, for example polymers (e.g., PLGA and/or pNIPAM),metals (e.g., gold), semiconductors, and the like. In one illustrativeembodiment, the core/shell nanoparticle may have a core comprising, forexample, iron oxide and a shell comprising gold. In one illustrativeembodiment, the core/shell nanoparticle may have a core comprising, forexample, PLGA and a shell comprising pNIPAM.

In various embodiments, the nanoparticles described herein can have atleast one dimension of about 1 nm to about 700 nm, about 1 nm to about500 nm, about 1 nm to about 250 nm, about 100 nm to about 700 nm, about100 nm to about 500 nm, about 100 to about 250 nm, about 250 to about700 nm, about 250 to about 500 nm, or about 500 nm to about 700 nm. Invarious embodiments, the nanoparticles described herein can have atleast one dimension of about 1 nm to about 100 nm, about 1 nm to about10 nm, about 1 nm to about 20 nm, about 1 nm to about 30 nm, about 1 nmto about 40 nm, about 1 nm to about 50 nm, about 1 nm to about 60 nm,about 1 nm to about 70 nm, about 1 nm to about 80 nm, or about 1 nm toabout 90 nm. In various embodiments, the nanoparticles described hereincan have at least one dimension of about 30 nm to about 100 nm, about 40nm to about 100 nm, about 50 nm to about 100 nm, about 60 nm to about100 nm, about 20 nm to about 80 nm, about 30 nm to about 50 nm, or about20 nm to about 50 nm. These various nanoparticle size ranges are alsocontemplated where the term “about” is not included.

In one illustrative embodiment, the nanoparticle is coupled to a“stabilizer.” A stabilizer, for example, can inhibit or can preventaggregation of the nanoparticles. Illustrative examples of stabilizersinclude, but are not limited to, a polyethylene glycol (PEG), a dextran,a peptide, an alkane-thiol, and an oligonucleotide-thiol. Peptidestabilizers, for example, those having the amino acid sequence CALNN(SEQ ID NO: 18) and its derivatives, are described by Levi at el. (2004)J. Am. Chem. Soc. 126: 10076-10084, incorporated herein by reference.Alkane-thiols and oligonucleotide-thiols are described by Jans et al.(2010) Nanotechnology 21: 1-8 and Cardenas et al. (2006) Langmuir 22:3294-3299, respectively, each of which is incorporated herein byreference. The molecular weight of the stabilizer may be variedaccording to the size of the coupled polypeptides to effectivelymaintain stability of the nanoparticle with minimal interference inspecific binding of the polypeptide to its target.

In one illustrative embodiment described herein are compositionscomprising at least one collagen binding polypeptide coupled to ananoparticle, wherein the polypeptide contains from 7 to 40 amino acidresidues and wherein the polypeptide does not form a triple helix. Atriple helix, for example, a collagen triple helix, is a quaternarystructure containing three left-handed helices twisted together.Illustratively, a peptide with repeating units of a sequence of Z-Z-G,wherein Z is any amino acid and G is glycine, is a typical peptide motifthat forms a triple helix. Specifically, collagen mimetics, such as, forexample, a peptide comprising repeating units ofProline-Hydroxyproline-Glycine, are exemplary of a peptide that forms atriple helix.

As used in accordance with this invention, “nanoparticle coupledcollagen binding peptides” refers to one or more collagen bindingpeptides coupled to one or more nanoparticles. In one illustrativeaspect, a nanoparticle is coupled to a collagen binding peptide thatbinds a single type of collagen (for example, such as collagen type I,or type II, or type III, or type IV, or type V to type XXIX). In anotherillustrative aspect, a nanoparticle is coupled to a collagen bindingpeptide that binds multiple types of collagen (i.e. is not specific fora single collagen type). In another illustrative aspect, a nanoparticleis coupled to multiple copies of the same collagen binding peptide. Inanother illustrative aspect, a nanoparticle is coupled to one or morecollagen binding peptides that differ in their specificity for bindingto collagen types.

In various illustrative embodiments, the collagen binding peptides maybind to any type of collagen, including collagen types I to XXIX, aloneor in any combination, for example, collagen types I, II (e.g., WYRGRLC(SEQ ID NO: 19) and WYRGRLGC (SEQ ID NO: 20)), III, and/or IV. Invarious illustrative aspects, the composition comprising collagenbinding peptides coupled to a nanoparticle comprises collagen bindingpeptides of about 7 to about 40 amino acids. In one illustrative aspect,the composition comprising collagen binding peptides coupled to ananoparticle comprises collagen binding peptides of about 7 to about 35amino acids, or from about 7 to about 30 amino acids, from about 7 toabout 25 amino acids, from about 7 to about 22 amino acids, from about 7to about 18 amino acids, from about 7 to about 15 amino acids, fromabout 9 to about 40 amino acids, from about 9 to about 18 amino acids,from about 15 to about 30 amino acids, or from about 15 to about 25amino acids. In various illustrative aspects, the composition comprisingcollagen binding peptides coupled to a nanoparticle comprises collagenbinding peptides of about 7 to about 50 or about 60 amino acids.

These various peptide amino acid ranges are also contemplated where theterm “about” is not included. For example, the composition comprisingcollagen binding peptides coupled to a nanoparticle can comprisecollagen binding peptides of 7 amino acids to 20 amino acids, 7 aminoacids to 35 amino acids, or from 7 amino acids to 30 amino acids, from 7amino acids to 25 amino acids, from 7 amino acids to 22 amino acids,from 7 amino acids to 18 amino acids, from 7 amino acids to 15 aminoacids, from 9 amino acids to 40 amino acids, from 9 amino acids to 18amino acids, from 15 amino acids to 30 amino acids, or from 15 aminoacids to 25 amino acids.

In some embodiments, the collagen binding peptides have homology to theamino acid sequence of a small leucine-rich proteoglycan or a plateletreceptor sequence. In various embodiments the synthetic peptidecomprises an amino acid sequence selected from the group consisting ofRRANAALKAGELYKSILY (SEQ ID NO: 1), RRANAALKAGELYKCILY (SEQ ID NO: 2),SYIRIADTNIT (SEQ ID NO: 5), TKKTLRT (SEQ ID NO: 6), SQNPVQP (SEQ ID NO:7), RLDGNEIKR (SEQ ID NO: 8), KELNVYT (SEQ ID NO: 9), KLWVLPK (SEQ IDNO: 10), CQDSETRTFY (SEQ ID NO: 11), AHEEISTTNEGVMGC (SEQ ID NO: 21),RLDGNEIKRGC (SEQ ID NO: 22), TKKTLRTGC (SEQ ID NO: 23),GLRSKSKKFRRPDIQYPDATDEDITSHMGC (SEQ ID NO: 24), SQNPVQPGC (SEQ ID NO:25), SYIRIADTNITGC (SEQ ID NO: 26), SYIRIADTNIT (SEQ ID NO: 5),KELNLVYTGC (SEQ ID NO: 27), GSITTIDVPWNV (SEQ ID NO: 14),NGVFKYRPRYFLYKHAYFYPPLKRFPVQ (SEQ ID NO: 15), GELYKSILY (SEQ ID NO: 3),GELYKCILY (SEQ ID NO: 4), NGVFKYRPRYFLYKHAYFYPPLKRFPVQGC (SEQ ID NO:28), GSITTIDVPWNVGC (SEQ ID NO: 29), RRANAALKAGELYKSILYGC (SEQ ID NO:30), GELYKSILYGC (SEQ ID NO: 31), GCGGELYKSILY (SEQ ID NO: 32), WYRGRLC(SEQ ID NO: 19), WYRGRLGC (SEQ ID NO: 20), and an amino acid sequencewith 80%, 85%, 90%, 95%, or 98% homology to any of these twenty eightamino acid sequences. The collagen binding peptide can also be anypeptide of about 7 amino acids to about 40 amino acids, or 7 amino acidsto 40 amino acids, selected from peptides that have collagen-bindingactivity and that are 80%, 85%, 90%, 95%, 98%, or 100% homologous withthe collagen-binding domain(s) of the von Willebrand factor or aplatelet collagen receptor as described in Chiang, et al., J. Biol.Chem. 277: 34896-34901 (2002), Huizinga, et al., Structure 5: 1147-1156(1997), Romijn, et al., J. Biol. Chem. 278: 15035-15039 (2003), andChiang, et al., Cardio. & Haemato. Disorders-Drug Targets 7: 71-75(2007), each incorporated herein by reference.

In other illustrative embodiments, the collagen-binding peptide can beselected from the group consisting of RRANAALKAGELYKSILY (SEQ ID NO: 1),RRANAALKAGELYKCILY (SEQ ID NO: 2), SYIRIADTNIT (SEQ ID NO: 5), TKKTLRT(SEQ ID NO: 6), SQNPVQP (SEQ ID NO: 7), and RLDGNEIKR (SEQ ID NO: 8),can comprise the amino acid sequence GELYKXILY, wherein X is serine,cysteine, or threonine (SEQ ID NO: 16), can be RRANAALKAGELYKSILY (SEQID NO: 1), or can be RRANAALKAGELYKCILY (SEQ ID NO: 2).

Conservative and/or nonconservative amino acid substitutions arecontemplated for all of the above-described peptides. Non-conservativesubstitutions are possible provided that these do not excessively affectthe collagen binding activity of the peptide.

As is well-known in the art, a “conservative substitution” of an aminoacid or a “conservative substitution variant” of a peptide refers to anamino acid substitution which maintains: 1) the secondary structure ofthe peptide; 2) the charge or hydrophobicity of the amino acid; and 3)the bulkiness of the side chain or any one or more of thesecharacteristics. Illustratively, the well-known terminologies“hydrophilic residues” relate to serine or threonine. “Hydrophobicresidues” refer to leucine, isoleucine, phenylalanine, valine oralanine, or the like. “Positively charged residues” relate to lysine,arginine, ornithine, or histidine. “Negatively charged residues” referto aspartic acid or glutamic acid. Residues having “bulky side chains”refer to phenylalanine, tryptophan or tyrosine, or the like. A list ofillustrative conservative amino acid substitutions is given in TABLE 1.

TABLE 1 For Amino Acid Replace With Alanine D-Ala, Gly, Aib, β-Ala,L-Cys, D-Cys Arginine D-Arg, Lys, D-Lys, Orn D-Orn Asparagine D-Asn,Asp, D-Asp, Glu, D-Glu Gln, D-Gln Aspartic Acid D-Asp, D-Asn, Asn, Glu,D-Glu, Gln, D-Gln Cysteine D-Cys, S-Me-Cys, Met, D-Met, Thr, D-ThrGlutamine D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp Glutamic Acid D-Glu,D-Asp, Asp, Asn, D-Asn, Gln, D-Gln Glycine Ala, D-Ala, Pro, D-Pro, Aib,β-Ala Isoleucine D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met Leucine Val,D-Val, Met, D-Met, D-Ile, D-Leu, Ile Lysine D-Lys, Arg, D-Arg, Orn,D-Orn Methionine D-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-ValPhenylalanine D-Phe, Tyr, D-Tyr, His, D-His, Trp, D-Trp Proline D-ProSerine D-Ser, Thr, D-Thr, allo-Thr, L-Cys, D-Cys Threonine D-Thr, Ser,D-Ser, allo-Thr, Met, D-Met, Val, D-Val Tyrosine D-Tyr, Phe, D-Phe, His,D-His, Trp, D-Trp Valine D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met

In various embodiments, the collagen binding peptide further comprises acysteine added to the amino terminus or the carboxy terminus of thepeptide. As used herein, the terms “amino terminus” or “carboxyterminus” refer to the first or last amino acid of the peptide.

In another embodiment, the added cysteine includes a spacer. In oneembodiment, the spacer comprises one or more glycines. Illustratively,the spacer and cysteine may comprise a dipeptide of GC or CG, or atripeptide of GCG. In one embodiment, wherein the cysteine is includedwith a spacer, the cysteine may then ultimately reside in the aminoterminal region or carboxy terminal region. As used herein, the terms“amino terminal region” or “carboxy terminal region” refer to positionsin the peptide that are 1, 2, 3, 4, or 5 positions from the aminoterminus or carboxy terminus.

In one embodiment, the collagen binding peptide is synthesized accordingto solid phase peptide synthesis protocols that are well-known bypersons of skill in the art. In one embodiment, a peptide precursor issynthesized on a solid support according to the well-known Fmocprotocol, cleaved from the support with trifluoroacetic acid andpurified by chromatography according to methods known to persons skilledin the art.

In another embodiment the synthetic peptide is synthesized utilizing themethods of biotechnology that are well known to persons skilled in theart. In one embodiment a DNA sequence that encodes the amino acidsequence information for the desired peptide is ligated by recombinantDNA techniques known to persons skilled in the art into an expressionplasmid (for example, a plasmid that incorporates an affinity tag foraffinity purification of the peptide), the plasmid is transfected into ahost organism for expression, and the peptide is then isolated from thehost organism or the growth medium according to methods known by personsskilled in the art (e.g., by affinity purification). Recombinant DNAtechnology methods are described in Sambrook et al., “Molecular Cloning:A Laboratory Manual”, 3rd Edition, Cold Spring Harbor Laboratory Press,(2001), incorporated herein by reference, and are well-known to theskilled artisan.

In any of the embodiments described herein, the nanoparticle coupledcollagen binding peptides can be administered alone or in combinationwith suitable pharmaceutical carriers or diluents. Diluent or carrieringredients used in the compositions containing collagen-bindingpeptides coupled to nanoparticles can be selected so that they do notdiminish the desired effects of the nanoparticle coupled collagenbinding peptides. Examples of suitable dosage forms include aqueoussolutions of the collagen-binding peptide coupled nanoparticles, forexample, a solution in isotonic saline, 5% glucose or other well-knownpharmaceutically acceptable liquid carriers such as alcohols, glycols,esters and amides.

“Carrier” is used herein to describe any ingredient other than theactive component(s) in a formulation. The choice of carrier will to alarge extent depend on factors such as the particular mode ofadministration, the effect of the carrier on solubility and stability,and the nature of the dosage form. In one illustrative aspect, thecarrier is a liquid carrier. In one illustrative aspect, the liquidcarrier is a pharmaceutically acceptable carrier.

“Pharmaceutically acceptable” as used in this application, for example,with reference to salts and formulation components such as carriers,includes “veterinarily acceptable”, and thus includes both human andanimal applications independently. For example, a “patient” as referredto herein can be a human patient or a veterinary patient, such as adomesticated animal (e.g., a pet).

Pharmaceutically acceptable salts, and common methodologies forpreparing pharmaceutically acceptable salts, are known in the art. See,e.g., P. Stahl, et al., HANDBOOK OF PHARMACEUTICAL SALTS: PROPERTIES,SELECTION AND USE, (VCHA/Wiley-VCH, 2002); S. M. Berge, et al.,“Pharmaceutical Salts,” Journal of Pharmaceutical Sciences, Vol. 66, No.1, January 1977. A preferred salt is the hydrochloride salt.

The compositions described herein and their salts may be formulated aspharmaceutical compositions for systemic administration. Suchpharmaceutical compositions and processes for making the same are knownin the art for both humans and non-human mammals. See, e.g., REMINGTON:THE SCIENCE AND PRACTICE OF PHARMACY, (1995) A. Gennaro, et al., eds.,19th ed., Mack Publishing Co. Additional active ingredients may beincluded in the composition containing a collagen binding peptidecoupled to a nanoparticle, or a salt thereof.

In one illustrative embodiment, pharmaceutical compositions for use witha composition comprising collagen binding peptides coupled tonanoparticles for parenteral administration comprise: a) apharmaceutically active amount of the nanoparticle coupled collagenbinding peptide; b) a pharmaceutically acceptable pH buffering agent toprovide a pH in the range of about pH 4.5 to about pH 9; c) an ionicstrength modifying agent in the concentration range of about 0 to about300 millimolar; and d) water soluble viscosity modifying agent in theconcentration range of about 0.25% to about 10% total formula weight orany combinations of a), b), c) and d) are provided.

In various illustrative embodiments, the pH buffering agents for use inthe compositions and methods herein described are those agents known tothe skilled artisan and include, for example, acetate, borate,carbonate, citrate, and phosphate buffers, as well as hydrochloric acid,sodium hydroxide, magnesium oxide, monopotassium phosphate, bicarbonate,ammonia, carbonic acid, hydrochloric acid, sodium citrate, citric acid,acetic acid, disodium hydrogen phosphate, borax, boric acid, sodiumhydroxide, diethyl barbituric acid, and proteins, as well as variousbiological buffers, for example, TAPS, Bicine, Tris, Tricine, HEPES,TES, MOPS, PIPES, cacodylate, or MES.

In another illustrative embodiment, the ionic strength modulating agentsinclude those agents known in the art, for example, glycerin, propyleneglycol, mannitol, glucose, dextrose, sorbitol, sodium chloride,potassium chloride, and other electrolytes.

Useful viscosity modulating agents include but are not limited to, ionicand non-ionic water soluble polymers; crosslinked acrylic acid polymerssuch as the “carbomer” family of polymers, e.g., carboxypolyalkylenesthat may be obtained commercially under the Carbopol® trademark;hydrophilic polymers such as polyethylene oxides,polyoxyethylene-polyoxypropylene copolymers, and polyvinylalcohol;cellulosic polymers and cellulosic polymer derivatives such ashydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropylmethylcellulose, hydroxypropyl methylcellulose phthalate, methylcellulose, carboxymethyl cellulose, and etherified cellulose; gums suchas tragacanth and xanthan gum; sodium alginate; gelatin, hyaluronic acidand salts thereof, chitosans, gellans or any combination thereof.Typically, non-acidic viscosity enhancing agents, such as a neutral or abasic agent are employed in order to facilitate achieving the desired pHof the formulation.

In one illustrative aspect, parenteral formulations may be suitablyformulated as a sterile non-aqueous solution or as a dried form to beused in conjunction with a suitable vehicle such as sterile,pyrogen-free water. The preparation of parenteral formulations understerile conditions, for example, by lyophilization, may readily beaccomplished using standard pharmaceutical techniques well known tothose skilled in the art.

In one embodiment, the solubility of the nanoparticle coupled collagenbinding polypeptides used in the preparation of a parenteral formulationmay be increased by the use of appropriate formulation techniques, suchas the incorporation of solubility-enhancing agents.

In various embodiments, formulations for parenteral administration maybe formulated to be for immediate and/or modified release. Modifiedrelease formulations include delayed, sustained, pulsed, controlled,targeted and programmed release formulations. Thus, a nanoparticlecoupled collagen binding peptide may be formulated as a solid,semi-solid, or thixotropic liquid for administration as an implanteddepot providing modified release of the active compound.

In other embodiments, nanoparticle coupled collagen binding peptides andcompositions containing them can be administered topically. A variety ofdose forms and bases can be applied to the topical preparations, such asan ointment, cream, gel, gel ointment, plaster (e.g. cataplasm,poultice), solution, powders, and the like. These preparations may beprepared by any conventional method with conventional pharmaceuticallyacceptable carriers or diluents as described below.

For example, vaseline, higher alcohols, beeswax, vegetable oils,polyethylene glycol, etc. can be used. In the preparation of a creamformulation, fats and oils, waxes, higher fatty acids, higher alcohols,fatty acid esters, purified water, emulsifying agents etc. can be used.In the preparation of gel formulations, conventional gelling materialssuch as polyacrylates (e.g. sodium polyacrylate), hydroxypropylcellulose, hydroxypropyl methyl cellulose, polyvinyl alcohol,polyvinylpyrrolidone, purified water, lower alcohols, polyhydricalcohols, polyethylene glycol, and the like are used. In the preparationof a gel ointment preparation, an emulsifying agent (preferably nonionicsurfactants), an oily substance (e.g. liquid paraffin, triglycerides,and the like), etc. are used in addition to the gelling materials asmentioned above. A plaster such as cataplasm or poultice can be preparedby spreading a gel preparation as mentioned above onto a support (e.g.fabrics, non-woven fabrics). In addition to the above-mentionedingredients, paraffins, squalane, lanolin, cholesterol esters, higherfatty acid esters, and the like may optionally be used. Moreover,antioxidants such as BHA, BHT, propyl gallate, pyrogallol, tocopherol,etc. may also be incorporated. In addition to the above-mentionedpreparations and components, there may optionally be used any otherconventional formulations for incorporation with any other additives.

In various embodiments, the dosage of the nanoparticle coupled collagenbinding peptides can vary significantly depending on the patientcondition, the disease state being treated (e.g., arthritis) or theimaging technique being used, the route of administration and tissuedistribution, and the possibility of co-usage of other therapeutictreatments or imaging agents. The effective amount to be administered toa patient is based on body surface area, patient weight or mass, andphysician assessment of patient condition.

Suitable dosages of the nanoparticle coupled collagen binding peptidescan be determined by standard methods, for example by establishingdose-response curves in laboratory animal models or in humans inclinical trials. Illustratively, suitable dosages of nanoparticlecoupled collagen binding peptides (administered in a single bolus orover time) include from about 1 pg/kg to about 10 μg/kg, from about 1pg/kg to about 1 μg/kg, from about 100 pg/kg to about 500 ng/kg, fromabout 1 pg/kg to about 1 ng/kg, from about 1 pg/kg to about 500 pg/kg,from about 100 pg/kg to about 500 ng/kg, from about 100 pg/kg to about100 ng/kg, from about 1 ng/kg to about 10 mg/kg, from about 1 ng/kg to 1mg/kg, from about 1 ng/kg to about 1 μg/kg, from about 1 ng/kg to about500 ng/kg, from about 100 ng/kg to about 500 μg/kg, from about 100 ng/kgto about 100 μg/kg, from about 1 μg/kg to about 500 μg/kg, or from about1 μg/kg to about 100 μg/kg. In each of these embodiments, dose/kg refersto the dose per kilogram of a patient's or animal's mass or body weight.

In another illustrative aspect, any of the above described compositionembodiments can be used in a method for imaging a collagenous matrix.For example, in one embodiment, a composition for use in imaging acollagenous matrix is provided. The composition comprises at least onecollagen binding polypeptide coupled to a nanoparticle, wherein thepolypeptide contains from 7 amino acids to 40 amino acids and whereinthe polypeptide does not form a triple helix. In another illustrativeembodiment, a method for imaging a collagenous matrix is provided. Themethod comprises the steps of contacting a collagenous matrix with acomposition comprising at least one collagen binding polypeptide coupledto a nanoparticle, wherein the polypeptide contains from 7 amino acidsto 40 amino acids and wherein the polypeptide does not form a triplehelix, and imaging the collagenous matrix.

The collagen-binding gold nanoparticles described herein, for example,are useful for a variety of biomedical imaging techniques including SEM,TEM, confocal microscopy, MRI, and photoacoustic imaging. Further,radiopaque nanoparticles that are coupled with collagen binding proteinscan be used in radiologic imaging such as CT scan or X-ray. The imagingmethods in which the collagen-binding peptides coupled to nanoparticlesare useful include both in vitro imaging methods and imaging methodsapplicable to a human or a veterinary patient. For example, any tissuecan be imaged to which a collagen binding polypeptide coupled to ananoparticle can be targeted.

Exemplary of tissues containing collagen that may be imaged inaccordance with the methods and compositions described herein includesubmucosa tissues (e.g., intestinal, urinary bladder tissue, and stomachtissue), pericardial tissue, skin tissue, bone, cartilage, tendon, otherconnective tissues of any animal, and any other collagen containingtissues of an animal.

In another embodiment, the compositions described herein can be used fortargeted drug delivery (e.g., to target drugs to tissues using thecollagen binding polypeptide as the targeting agent). For example, thecompositions can be used for targeted delivery of drugs to specifictissues, and further to increase solubility of drugs under physiologicalconditions. Solubility limits have prevented the use of numerouseffective drugs. However, solubility problems can be overcome by the useof nanoparticles, in which the insoluble compound is encapsulated. Inother embodiments, the nanoparticles can be used to control drug releaseusing engineered nanoparticles with specifically designed geometries anddegradation profiles, making possible the release of effective drugdoses over long periods of time.

Peptides can be coupled to nanoparticles by employing a variety ofchemistries, for example, such as those described in BioconjugateTechniques (Greg T. Hermanson, Academic Press; 2 edition (May 2, 2008)),incorporated herein by reference. Illustratively, a collagen bindingsequence, for example, RRANAALKAGELYKSILY (SEQ ID NO: 1) may be modifiedsuch that it contains a glycine spacer followed by a cysteine at thecarboxy terminus yielding the sequence RRANAALKAGELYKSILYGC (SEQ ID NO:30).

The sulfhydryl group contained in the cysteine may then be used tocouple the peptide to gold on the gold nanoparticle. PEG-SH is firstcoupled to the gold nanoparticles through its sulfhydryl group, thenexcess PEG-SH is removed by centrifugation and reconstituting theparticles in PBS buffer. The modified collagen-binding peptide is thenincubated with PEG functionalized gold nanoparticles in excess, suchthat the peptide couples to the gold.

Other chemistry techniques may be used for coupling peptides tonanoparticles depending on the peptide sequence. For example, if thecollagen-binding peptide sequence contains an internal cysteine whichcannot be modified, it may be possible to use an amine group forcoupling. Crosslinking molecules may be used for such coupling. Forexample, a heterobifunctional crosslinker commercially available fromThermo Scientific that couples amines to sulfhydryl groups may be used.

Example 1 Nanoparticle Functionalization

Gold nanoparticles (NP) of 13 nm size were prepared as previouslydescribed and stabilized with citrate until further functionalization(Jeong et al. (2008) Langmuir (16):8794-8800; Storhoff et al. (1998)JAGS 120(9): 1959-1964). Peptide (SILY_(biotin) (SEQ ID NO: 33)) waspurchased from Genscript (Piscataway, N.J.). Monofunctional PEG-SH (1kDa, 2 kDa, or 5 kDa) was purchased from Layasan Bio (Arab, AL).Streptavidin-HRP and color evolving solutions were purchased from R&DSystems (Minneapolis, Minn.). All other reagents were purchased from VWR(Radnor, Pa.).

Gold nanoparticles were functionalized following a previously describedmethod (Liu et al. (2007) Analytical Chem. 79(6):2221-2229).Monofunctional PEG-SH was dissolved in 1×PBS pH 7.4 to a finalconcentration of 0.01 mg/mL. PEG-SH was added to 1 mL of 13 nm NP incitrate to a final molar ratio of 2,500:1 (PEG:NP) and allowed to reactfor 1 hour at room temperature. PEG functionalized GNP (PEG-NP) was thenpelleted by centrifuging at 15,000 RPM for 15 minutes. The supernatantwas removed and particles were reconstituted in 1×PBS pH 7.4. Theprocess was repeated 2 more times to remove excess PEG.

Biotin labeled peptide (SILY_(biotin) (SEQ ID NO: 33)) was dissolved inultrapure water to a final concentration of 2 mg/mL, and was added topurified PEG-NP at a molar ratio of 1,000:1 (SILY_(biotin):PEG-NP(‘SILY_(biotin)’ disclosed as SEQ ID NO: 33)). Peptide coupling to theNP was reacted overnight at room temperature. Excess peptide was removedby centrifugation as described for PEG purification and was repeated 3times. The final product PEG-NP-SILY (‘SILY’ disclosed as SEQ ID NO: 17)was then brought up to a stock concentration of approximately 10 nM andwas used within 2 days of synthesis.

Example 2 Collagen-Binding of Nanoparticles

Fibrillar collagen (Chronolog, Havertown, Pa.) was diluted in isotonicglucose to 0.1 mg/mL and was coated onto 96-well plates (Greiner)overnight at 4 C. Control wells were coated with 1% BSA in 1×PBS pH 7.4at room temperature for 1 hour. All wells were then rinsed with 1×PBS pH7.4 to remove unbound collagen or BSA. PEG-NP-SILY (‘SILY’ disclosed asSEQ ID NO: 17) was then incubated on the surfaces at varyingconcentrations and allowed to bind for 30 min at room temperature. Wellswere then rinsed with 1×PBS 3 times to remove unbound PEG-NP-SILY(‘SILY’ disclosed as SEQ ID NO: 17). Bound PEG-NP-SILY (‘SILY’ disclosedas SEQ ID NO: 17) was then detected by probing for biotin usingStreptavidin-HRP following manufacturers protocol.

Stability of PEG-NP-SILY (‘SILY’ Disclosed as SEQ ID NO: 17)

Gold nanoparticles will aggregate in solution unless stabilized, such asin citrate buffer. In 1×PBS gold nanoparticles immediately precipitate,and conjugating the SILY peptide (SEQ ID NO: 17) to nanoparticles alsoresults in aggregation in PBS. PEG is therefore used to stabilize thenanoparticles. Sequential coupling of PEG following by SILY (SEQ ID NO:17) was previously shown to be an effective method of functionalizingthe NP with peptide. Here, the same method was employed to couple SILY(SEQ ID NO: 17).

Binding to Type I Collagen

The molecular weight of PEG plays a significant role in the function ofPEG-NP-SILY (‘SILY’ disclosed as SEQ ID NO: 17) as shown in FIG. 1.While PEG-1 kDa allows for binding to collagen, there is significantnonspecific binding to the BSA surface and was thus not used (data notshown). Conversely, PEG-2 kDa and PEG-5 kDa result in specific bindingof the functionalized NP to collagen with minimal binding to the BSAsurface. Increased binding was observed with PEG-2 kDa compared withPEG-5 kDa. This result is due to the size of the peptide, in which SILY(SEQ ID NO: 17) is 2197 Da, and is thus excluded to some degree with 5kDa PEG. The optimum molecular weight of PEG is thus from 2 kDa to 5kDa, and could vary depending on the peptide sequence.

NP functionalized with 2 kDa PEG showed increased binding to collagencompared to 5 kDa PEG. This is due to the length of the peptide SILY(SEQ ID NO: 17), which is 2197 Da and is masked in part by the highermolecular weight PEG. Minimal nonspecific binding to BSA was observed inboth functionalized nanoparticles (FIG. 2).

Example 3 Visualization of SILY (‘SILY’ Disclosed as SEQ ID NO: 17)Functionalized Gold Nanoparticles on Rat Tail Tendon Collagen

Gold nanoparticles of 60 nm diameter were purchased from Cytodiagnostics(Burlington, ON). Particles were functionalized with PEG-thiol followedby free SILY_(biotin) (SEQ ID NO: 33) peptide yielding GNP-SILY (‘SILY’disclosed as SEQ ID NO: 17). Rat tail tendons were harvested fromSprague-Dawley rats and rinsed in 1×PBS immediately prior to testing.Plates (96-well) were coated with 1% BSA to prevent nonspecific binding,and rinsed 3× to remove unbound BSA. Tendons were then incubated for 15min at room temperature in 100 μL/well of approximately 0.01 nM GNP-SILY(‘SILY’ disclosed as SEQ ID NO: 17) in 1×PBS pH 7.4 or in controlnanoparticles which were functionalized with PEG only (GNP-PEG). Tendonswere then rinsed in 3× in 200 μL 1×PBS for 5 minutes per rinse to washof unbound nanoparticles. Samples were fixed in 2.5% glutaraldehyde in1×PBS pH 7.4 at 4° C. for 2 hours followed by rinsing and dehydration ingraded ethanol up to 100% and air dried.

Tendons were platinum sputter coated and examined by SEM. Representativeimages shown in FIG. 3 demonstrate specific binding of GNP-SILY (‘SILY’disclosed as SEQ ID NO: 17) to collagen in tendons as noted by the 60 nmspheres. In contrast, control tendon incubated with GNP-PEG did notcontain any nanoparticles. The number of particles bound to the tendonwas rather sparse and is most likely due to the very low concentrationof nanoparticles (0.01 nM). To increase particle binding, nanoparticlesmay be further concentrated prior to incubating tendons.

Example 4 Core+Shell Nanoparticle Synthesis

Synthesis of the core+shell nanoparticles occurred in two steps:synthesis of the Poly(lactic-co-glycolic acid) (PLGA) cores followed bythe addition of the poly(N-isopropylacrylamide) (p NIP AM) shell (FIG.4). PLGA cores were synthesized using a single emulsion technique.Briefly, 5 g of poly(dl-lactide/glycolide) 50:50 (PLGA; PolysciencesInc.) were dissolved in 5 mL of dichloromethane (DCM; Sigma-Aldrich),added to 20 mL of 5% polyvinyl alcohol (PVA; Alfa Aesar), andhomogenized for 30s using a probe sonicator (Branson Sonifier 450) togenerate a single emulsion. The emulsion was added to 100 mL of rapidlystirred distilled water and left overnight to allow for full evaporationof the DCM. The PLGA nanoparticles were further purified viacentrifugation washes with distilled water. Any clumps of PLGAnanoparticles that remained after centrifugation were disrupted usingbrief sonication.

The PLGA cores were encapsulated in pNIPAM shells using aqueous freeradical precipitation polymerization under a nitrogen atmosphere.Briefly, 0.27 g N-isopropylacrylamide [2.385 mmol] (Polysciences Inc.),0.021 g N—N′-methylene bisacrylamide [0.136 mmol] (Fluka), 0.012 gsodium dodecyl sulfate [0.042 mmol] (Sigma Aldrich), and 0.015 gammonium persulfate [0.066 mmol] (Sigma Aldrich) were dissolved in 30 mLof distilled water and purged of oxygen by nitrogen bubbling. Fornanoparticle targeting, either 1.67 μl [0.024 mmol or 1 mol %] or 8.35μl [0.121 mmol or 5 mol %] of acrylic acid (AAc; Alfa Aesar) wereincluded. 20 mL of the PLGA nanoparticle cores were added to a 250 mLthree-neck round bottom flask and equilibrated to 70° C. for 20 minutesunder nitrogen with stirring. 10 mL of the shell solution was added tothe 70° C. equilibrated PLGA nanoparticle cores and allowed topolymerize. Additional 5 mL aliquots of shell solution were added 30,50, 70, and 90 minutes after the initial polymerization. Polymerizationcontinued for 6 hours after the final addition of shell solution.Purification was achieved through dialysis of the PLGA core+pNIPAM shellnanoparticles against distilled water for 7 days in 15,000 MWCO dialysistubing (Spectrum Laboratories, Inc.). Centrifugation washes wereperformed in order to further isolate the core+shell nanoparticles. Anyclumps of core+shell nanoparticles post centrifugation were dispersedusing brief sonication. A portion of the samples were lyophilized andthen rehydrated in distilled water.

Example 5 Transmission Electron Microscopy (TEM) Characterization

Images were taken of the pre- and post-lyophilized nanoparticle samplesby staining them with 2% uranyl acetate. The stained nanoparticles wereplaced on a glow-discharged 400 mesh coated with formvar+carbon film,and placed in a Philips CM-100 TEM where images were captured on KodakSO-163 electron image film.

Lyophilization is an effective way to prevent the release oftherapeutics loaded into PLGA nanoparticles during long-term storage.TEM imaging was used to visually confirm that well-defined sphericalPLGA and PLGA core+pNIPAM shell nanoparticles were successfullysynthesized pre-lyophilization (FIG. 5). TEM imaging showed that postlyophilization the PLGA nanoparticles form aggregates (FIG. 5). Thisaggregation resulted in an inability to measure diameter and ζ-potentialof post-lyophilized PLGA nanoparticles. In contrast, encapsulation ofPLGA nanoparticles with pNIPAM shells prevented aggregation of the PLGAnanoparticles following lyophilization, further confirming that thepNIPAM shell fully encapsulated the PLGA core. Furthermore,lyophilization of the core+shell nanoparticles did not affect the sizeor ζ-potential of the particles at 25° C. or 37° C. (Table 1).

Example 6 Nanoparticle Sizing and Zeta Potential

Measurements were taken with a Malvern Zetasizer Nano Z590. Pre- andpost-lyophilized nanoparticles were suspended in distilled water andanalyzed for particle size in polystyrene cuvettes at 25° C. and 37° C.Temperature sweeps were performed by varying temperature from 20° C. to50° C. to 20° C. in 1° increments with measurement of particle size witheach change in degree. Disposable Malvern ζ-potential cuvettes were usedto acquire ζ-potential measurements at 25° C. and 37° C. After makingany change in temperature, nanoparticle samples were allowed toequilibrate for five minutes before any sizing or zeta measurements weremade.

Verification of successful encapsulation of the PLGA cores with thepNIPAM shells was initially achieved using dynamic light scattering tomeasure the size of the nanoparticles before and after the addition ofthe shell to determine the change in nanoparticle diameter. The diameterof the core+shell nanoparticles (Table 1) increased post addition of thepNIPAM shell suggesting successful encapsulation. pNIPAM shell thicknessranged from ˜100 nm to ˜200 nm at 25° C., with an increase in shellthickness corresponding to an increase in the mole percent of acrylicacid incorporated into the pNIPAM.

Additional verification of the successful encapsulation of the pNIPAMshell was provided by confirming a phase transition when the temperaturewas raised above its lower critical solution temperature (LCST). Allcore+shell nanoparticles had reduced diameters at 37° C. compared to 25°C. (Table 1).

To assess colloidal stability of the core+shell nanoparticles above andbelow the phase transition temperature, the ζ-potentials were measured.Typically, nanoparticle ζ-potentials above 30 mV or below −30 mV areconsidered stable.

TABLE 1 Characterization of Various Nanoparticle Samples. Particle ZetaShell Sample Lyophili- T Diameter Potential Thickness Name zation? (°C.) (nm) (mV)^(a) (nm)^(b) PLGA No 25 392.3 ± 13.9 −32.1 ± 2.0 NA Core37 377.2 ± 20.2 −22.5 ± 3.4 PLGA No 25 605.3 ± 10.8 −28.3 ± 4.4 106.5 ±5.4  Core + 37 403.8 ± 7.1  −25.0 ± 2.5 13.3 ± 3.6 pNIPAM Yes 25 610.2 ±14.2 −32.1 ± 0.9 109.0 ± 7.1  Shell 0% 37 414.5 ± 7.9  −24.3 ± 1.8 18.6± 3.9 AAc PLGA No 25 659.1 ± 6.7  −28.0 ± 6.0 133.4 ± 3.4  Core + 37428.0 ± 9.5  −22.7 ± 2.7 25.4 ± 4.7 pNIPAM Yes 25 681.7 ± 20.9 −30.8 ±5.1 144.7 ± 10.4 Shell 1% 37 447.0 ± 13.5 −20.5 ± 0.7 34.9 ± 6.8 AAcPLGA No 25 748.5 ± 17.1 −34.9 ± 2.5 178.1 ± 8.5  Core + 37 534.1 ± 14.8−25.1 ± 3.1 78.4 ± 7.4 pNIPAM Yes 25 818.3 ± 3.9  −34.6 ± 0.2 213.0 ±2.0  Shell 5% 37 575.9 ± 16.6 −25.2 ± 2.1 99.4 ± 8.3 AAc ^(a)pH of allsamples was between 5 and 6. ^(b)Determined by subtracting correspondingcore from core + shell sample and dividing by 2.

To further assess the response of the core+shell nanoparticles totemperature-based environmental stimuli, dynamic light scattering wasused to measure the diameter of the core+shell nanoparticles as theywere exposed to a dynamic range of temperatures from 20° C. to 50° C. to20° C. The core+shell nanoparticles readily responded within thistemperature range with all core+shell nanoparticle types decreasing indiameter as the temperature was raised above the LCST (FIG. 6). Thisresponse was reversible as the nanoparticles returned to their originaldiameter when the temperature was lowered back below the LCST.Additionally, the LCST of the pNIPAM shell was tuned by modifying theamount of acrylic acid that was incorporated as a co-monomer. As moreacrylic acid was incorporated, the LCST of pNIPAM increased (FIG. 6).The core+shell nanoparticles with 0 mol % acrylic acid exhibited an LCSTat ˜31-32° C., while the 1 mol % acrylic acid had an LCST at ˜33-34° C.,and the core+shell nanoparticles with 5% acrylic acid had an LCST at˜35° C. (FIG. 6).

Example 7 Peptide Synthesis and Purification

The collagen type II binding peptide single amino acid sequenceconsisting of WYRGRLGC (SEQ ID NO: 20) was used. The peptide wassynthesized at a 0.4 mmol scale on Knorr-amide resin (Synbiosci Corp.)using standard FMOC (9-fluorenylmethyloxycarbonyl) chemistry. Twodifferent chemistries were used to couple each amino acid (SynbiosciCorp). The first coupling reagents consisted of N-hydroxybenzotriazole(HOBt; Synbiosci) and N,N′ diisopropylcarbodiimide (DIC; Sigma-Aldrich)and the second coupling reagents were0-Benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate(HBTU; Synbiosci) and lutidine (Sigma-Aldrich). Following synthesis, thepeptide was cleaved from the resin with 95% trifluoroacetic acid(Sigma-Aldrich), 2.5% water, 1.25% triisopropylsilane (Sigma-Aldrich),and 1.25% ethanedithiol (Sigma-Aldrich), precipitated in cold ether, andrecovered by centrifugation. The peptide was purified with anacetonitrile gradient on an AKTA Explorer FPLC (GE Healthcare) equippedwith a 22/250 C18 reversed phase column (Grace Davidson). Molecularweight was confirmed by time of flight MALDI mass spectrometry using a4800 Plus MALDI TOF/TOF Analyzer (Applied Biosystems). Theoreticalmolecular weight of WYRGRLC (SEQ ID NO: 19) was calculated to be 1009.1while the actual molecular weight was found to be 1009.58. Abiotinylated version of the peptide (biotin-WYRGRLC (SEQ ID NO: 34)) waspurchased from Genscript and its theoretical molecular weight wascalculated to be 1179.42 while its actual molecular weight was found tobe 1179.8.

Example 8 Modifying Core+Shell Nanoparticle with Targeting Moiety

Nanoparticle targeting was achieved through the addition of a collagenbinding peptide to the AAc groups on our core+shell nanoparticles usinga heterobifunctional crosslinker. Briefly, 0.4 mg of1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC;Thermo-Scientific) and 1.1 mg of N-hydroxylsulfosuccinimide (Sulfo-NHS;Thermo-Scientific) were added to 1 mg of core+shell nanoparticles for 15minutes in activation buffer (0.1M 2-(N-morpholino)ethanesulfonic acid(MES; Amresco) pH 6.0). Excess EDC and sulfo-NHS was removed by acentrifuge wash. The heterobifunctional crosslinker,N-[β-maleimidopropionic acid] hydrazide (BMPH; Thermo-Scientific) wasadded to the activated nanoparticles (0.1 mg for 1 mol % AAcnanoparticles or 0.3 mg for 5 mol % AAc nanoparticles) for 2 hours incoupling buffer (0.1M MES, pH 7.2). Excess BMPH was removed using gelfiltration chromatography (GFC) through an ÄKTA Purifier FPLC (GEHealthcare) with Bio-Scale Mini Bio-Gel columns packed withpolyacrylamide beads (Bio-Rad Laboratories). The collagen bindingpeptide (15% biotinylated) was added to the nanoparticles for 2 hours incoupling buffer. Excess peptide was removed via gel filtrationchromatography. Confirmation of peptide addition was performed using aflouraldehyde assay (Pierce), which reacts with free amines and astreptavidin color development assay which confirmed the presence of thebiotinylated peptide on the nanoparticle surface (data not shown).

The acrylic acid allowed the addition of a heterobifunctionalcrosslinker, e.g., BMPH, using EDC/NHS chemistry. The thiol basedcysteine in the collagen binding peptide was reacted with a maleimidebased BMPH. This chemistry can be applied to attach other targetingmoieties that contain a free thiol functional group.

Example 9 Collagen Binding Assay

Modified nanoparticles were tested for their ability to bind tocollagen. A 96-well plate (Greiner) was coated with collagen type IIfrom chicken sternum (Sigma) in 0.25% acetic acid at a concentration of0.5 mg/ml overnight. Following three washes, the plate was blocked with1% bovine serum albumin (BSA; SeraCare Life Systems) for 1 hour. Afterthree more washes, the collagen type II binding peptide modifiedcore+shell nanoparticles and unmodified controls were incubated in thecollagen type II coated plate for 1 hour. Following three more washes,streptavidin (R&D Systems) was diluted 200× in 1% BSA and incubated for20 minutes in the plate. After more washing to remove unboundstreptavidin, a color solution (R&D Systems) was added for 20 minutes.Sulfuric acid (Mallinckrodt Chemicals) was used to stop the reaction andabsorbance was read at 450 nm with a correction at 540 nm.

To assess whether incorporation of a collagen binding peptide wouldallow the core+shell nanoparticles to bind to collagen, a streptavidinELISA was used. The results from the assay indicate that core+shellnanoparticles modified with collagen type II binding peptide boundcollagen type II as compared to unmodified core+shell nanoparticlecontrols (FIG. 9; p<0.05, One-way ANOVA+Tukey post hoc test).Furthermore, the number of collagen type II binding peptide modifiedcore+shell nanoparticles able to bind to collagen type II was directlyrelated to the concentration of acrylic acid that was incorporated intothe pNIPAM shell.

Example 10 Cell Culture and Nanoparticle Biocompatibility

Immortalized human monocytes (THP-1, ATCC) were grown in RPMI 1640 withL-glutamine (Mediatech Inc) supplemented with 0.05 mM mercaptoethanol(Sigma-Aldrich), 10 mM HEPES (Mediatech Inc), 1 mM sodium pyruvate(Mediatech Inc), 10% fetal bovine serum (Hyclone), and 1%penicillin/streptomycin (Mediatech Inc). Cells were used between passagenumber 4 and 12 for all assays and maintained at 37° C. with 5% CO2.

The biocompatibility of the nanoparticles was assessed by measuringtoxicity and inflammation in THP-1 cells. Cells were seeded at a densityof 250,000 cells/ml in 96-well plates (Corning) and treated with 10ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich) for 48 hoursto induce differentiation, which was confirmed by the monocytes becomingadherent. Following a change of media, cells were treated with variousconcentrations of core+shell nanoparticles. Control samples received PBS(negative control) or 50 ng/ml lipopolysaccharide (LPS, Sigma-Aldrich)(positive control). After 24 hours, the media was collected for cytokineanalysis and an MTT-based assay was performed to determine cell toxicityusing the Aqueous One Proliferation Kit (Promega) according tomanufacturer's instructions. Briefly, 20 μl of reagent was addeddirectly to 100 μl of cells and media. After two hours of incubation inthe cell culture incubator, the absorbance was read at 490 nm with acorrection at 650 nm.

The biocompatibility of the core+shell nanoparticles was assessed byevaluating the toxicity and inflammatory response in an immortalizedhuman monocyte cell line, i.e., THP-1 cells. Monocytes have asignificant role in the perpetuation of osteo- and rheumatoid arthritis.The core+shell nanoparticles were not toxic at any of the concentrationstested as no significant reduction in THP1 proliferation was observed(FIG. 7). However, the core+shell nanoparticles with 5 mol % acrylicacid induced a significant increase in proliferation of the THP-1 cellsat concentrations of 2.5 and 5 mg/mL compared to all other treatments(One-way ANOVA p<0.05; Tukey post-hoc test). The ability of thecore+shell nanoparticles to elicit an inflammatory response wasdetermined by measuring TNF-α production by THP-1 cells using an ELISA.Similar to the PBS control, the core+shell nanoparticles did not elicitTNF-α production (FIG. 8; p>0.05 One-way ANOVA). The positive control,THP-1 cells treated with lipopolysaccharide, did induce TNF-α productionas expected.

Example 11 Cytokine Analysis

The ability of the particles to cause an inflammatory response wasdetermined by running conditioned cell media on a TNF-α ELISA(PeproTech) according to manufacturer instructions. Briefly, NuncMaxiSorp 96-well plates were coated with capture antibody overnight.After blocking for one hour with 1% bovine serum albumin (SeraLifesciences) in PBS, samples and standards were incubated for two hourswith gentle rotation. Following incubation with a detection antibody andan avidin-horse radish peroxidase conjugate, the samples were developedwith the addition of 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonicacid) (ABTS) liquid substrate (Sigma-Aldrich) and monitored at 405 nmwith a correction at 650 nm.

Example 12 Statistical Analysis

Data was analyzed for differences using a single factor ANOVA with aTukey post-hoc test. An α=0.05 was used for all analyses. Graphs aredepicted as mean±standard deviation.

1.-37. (canceled)
 38. A composition comprising a) a plurality ofnanoparticles wherein each nanoparticle is covalently bound to at leastone collagen binding polypeptide, wherein the polypeptide contains from7 amino acids to 40 amino acids and wherein the polypeptide does notform a triple helix and b) a drug.
 39. The composition of claim 38,wherein the drug is encapsulated into the nanoparticle.
 40. Thecomposition of claim 38 wherein the nanoparticle further comprises astabilizer.
 41. The composition of claim 40 wherein the stabilizer isselected from the group consisting of a polyethylene glycol (PEG), adextran, a peptide, an alkane-thiol, and an oligonucleotide-thiol. 42.The composition of claim 41 wherein the stabilizer is PEG.
 43. Thecomposition of claim 38 wherein the polypeptide contains from 9 aminoacids to 40 amino acids.
 44. The composition of claim 38 wherein thepolypeptide contains from 9 amino acids to 30 amino acids.
 45. Thecomposition of claim 38 wherein the polypeptide is selected from thegroup consisting of RRANAALKAGELYKSILY (SEQ ID NO: 1),RRANAALKAGELYKCILY (SEQ ID NO: 2), GELYKSILY (SEQ ID NO: 3), GELYKCILY(SEQ ID NO: 4), SYIRIADTNIT (SEQ ID NO: 5), TKKTLRT (SEQ ID NO: 6),SQNPVQP (SEQ ID NO: 7), RLDGNEIKR (SEQ ID NO: 8), KELNVYT (SEQ ID NO:9), KLWVLPK (SEQ ID NO: 10), CQDSETRTFY (SEQ ID NO: 11), AHEEISTTNEGVM(SEQ ID NO: 12), GLRSKSKKFRRPDIQYPDATDEDITSHM (SEQ ID NO: 13),GSITTIDVPWNV (SEQ ID NO: 14), and NGVFKYRPRYFLYKHAYFYPPLKRFPVQ (SEQ IDNO: 15).
 46. The composition of claim 38 wherein the polypeptide isselected from the group consisting of RRANAALKAGELYKSILY (SEQ ID NO: 1),RRANAALKAGELYKCILY (SEQ ID NO: 2), GELYKSILY (SEQ ID NO: 3), GELYKCILY(SEQ ID NO: 4), SYIRIADTNIT (SEQ ID NO: 5), TKKTLRT (SEQ ID NO: 6),SQNPVQP (SEQ ID NO: 7), and RLDGNEIKR (SEQ ID NO: 8).
 47. Thecomposition of claim 38 wherein the polypeptide comprises the amino acidsequence GELYKXILY, wherein X is serine, cysteine, or threonine (SEQ IDNO: 16).
 48. The composition of claim 38 wherein the polypeptide isRRANAALKAGELYKSILY (SEQ ID NO: 1).
 49. The composition of claim 38wherein the polypeptide is RRANAALKAGELYKCILY (SEQ ID NO: 2).
 50. Thecomposition of claim 49 further comprising a spacer.
 51. The compositionof claim 50 wherein the spacer comprises at least one glycine.
 52. Thecomposition of claim 38 further comprising a peptide sequence selectedfrom the group consisting of GC, CG, and GCG.
 53. The composition ofclaim 38 further comprising a carrier.
 54. The composition of claim 38wherein the nanoparticle is a core plus shell nanoparticle.
 55. A methodfor targeted delivery of a drug to a patient in need thereof, comprisingadministering to the patient a composition of claim 38.